Inhibition Mechanisms Of African Swine Fever Virus Protein On CGAS-STING-mediated Signaling Pathway

African swine fever virus(ASFV)is a large DNA virus that infects domestic pigs and European wild boars with morbidity and mortality rates up to 100%.Since the first case of African swine fever(ASF)reported in China in last August,this disease has been spread to 31 provinces.Due to the large genomic structure and complex immune evasion mechanisms,there is still no commercail vaccine for ASFV.Cyclic GMP-AMP synthase(cGAS)is a recently discovered cytosolic DNA pattern recognition receptor that plays important roles in activating host innate immunity and regulating adaptive immunity.Upon infection,cGAS recognizes viral DNA and catalyzes the formation of the second messenger 2'3'-cGAMP.2'3'-cGAMP binds to adaptor protein stimulator of interferon genes(STING)leading to its dimerization,activation and translocation.Activated STING recruits TBK1 and IRF3,and promotes the phosphorylation of IRF3 by TBK1.STING also activates NF-?B signaling,possibly through TBK1 and IKK?.Activated IRF3 and NF-?B transport to the nucleus,mediating transcriptional expression of type I interferons and pro-inflammatory cytokines.cGAS-STING-mediated antiviral response is one of research hotspots.The current study aims to find cGAS-STING signaling antagonists from ASFV proteins and to explore the related mechanisms,which may provide reference for the development of safe and effective ASFV vaccines.The contents of the study are as follows: 1.Immunosuppressive properties of multigene family proteins of ASFV Georgia 2007Previous studies have shown that certain ASFV MGF360 and MGF505 proteins are involved in inhibiting the expression of type I interferon and antagonizing the antiviral effect mediated by type I IFN.Therefore,we initially screened the immunosuppressive genes from 43 MGF genes of ASFV Georgia 2007 and found 11 genes which inhibited cGAS-STING-stimulated IFN-? or NF-?B promoters activity.Furthermore,we found that MGF360-12 L,MGF360-13 L and MGF505-2R can also inhibit the mRNA expression levels of IFN-? in PAM cells and PK-15 cells.Among them,MGF360-12 L had a relatively stronger inhibitory effect.2.Mechanisms of inhibiting cGAS-STING-mediated antiviral innate immune response by ASFV MGF360-12LResults of dual luciferase reporter assays showed that MGF360-12 L inhibited cGAS-STING-stimulated IFN-? and NF-?B promoters activation and inhibited TBK1 or IRF3-5D-mediated IFN-? and ISRE promoter activation.And the expression of exogenous TBK1 and IRF3-5D proteins were also inhibited when cotransfected with MGF360-12 L.In addition,MGF360-12 L significantly inhibited NF-?B promoter activation induced by TBK1 or IKK? overexpression,but did not inhibit NF-?B promoter activation induced by p65 overexpression;Using Western blot assay and qRT-PCR,we found that MGF360-12 L inhibited exogenous TBK1 and IKK? protein expression levels and inhibited the mRNA expression levels of TBK1 and IKK?.Besides,MGF360-12 L protein can inhibit IFN-?-mediated antiviral effects to promoted VSV-GFP proliferation.Further studies indicated that MGF360-12 L inhibited IFN-?-mediated JAK-STAT signaling pathway by reducing total protein level of IRF9.3.Mechanisms of inhibiting cGAS-STING-mediated antiviral natural immune response by ASFV DP96RThe ASFV DP96 R gene is one of ASFV virulence factors,yet its action mechanism remains unknown.In this study,we reported that DP96 R subverted type I IFN production mediated by cGAS-STING sensing pathway.DP96 R inhibited the cGAS/STING or TBK1 but not IRF3-5D mediated IFN-? and ISRE promoters activation.Furthermore,DP96 R selectively blocked the activation of NF-?B promoter induced by cGAS/STING,TBK1 or IKK? but not by overexpression of p65.Moreover,DP96 R inhibited phosphorylation of TBK1 stimulated by cGAS/STING overexpression and TBK1-induced antiviral response.Finally,truncated mutation analysis demonstrated that the region spanning amino acids 30 to 96 of DP96 R was responsible for the inhibitory activity.In conclusion,we preliminarily studied the mechanisms of inhibition of cGAS-STING signaling by ASFV immunosuppressive proteins MGF360-12 L and DP96 R.The results could provide reference for further elucidation of viral immune evasion mechanisms and development of safe and effective ASFV vaccines.